Green transgenic fluorescent ornamental fish

ABSTRACT

The present invention relates to transgenic green ornamental fish, as well as methods of making such fish by in vitro fertilization techniques. Also disclosed are methods of establishing a population of such transgenic fish and methods of providing them to the ornamental fish industry for the purpose of marketing.

The present application claims the priority benefit of U.S. provisionalapplication Nos. 61/676,004, filed Jul. 26, 2012, and 61/722,989, filedNov. 6, 2012, each of which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to transgenic fish, particularly green transgenicfish.

2. Description of Related Art

Transgenic technology involves the transfer of a foreign gene into ahost organism enabling the host to acquire a new and inheritable trait.Transgenic technology has many potential applications. For example, itcan be used to introduce a transgene into a fish in order to create newvarieties of fish. There are many ways of introducing a foreign geneinto fish, including: microinjection (e.g., Zhu et al., 1985; Du et al.,1992), electroporation (Powers et al., 1992), sperm-mediated genetransfer (Khoo et al., 1992; Sin et al., 1993), gene bombardment or genegun (Zelenin et al., 1991), liposome-mediated gene transfer (Szelei etal., 1994), and the direct injection of DNA into muscle tissue (Xu etal., 1999). The first transgenic fish report was published by Zhu etal., (1985) using a chimeric gene construct consisting of a mousemetallothionein gene promoter and a human growth hormone gene. Most ofthe early transgenic fish studies have concentrated on growth hormonegene transfer with an aim of generating fast growing fish. While amajority of early attempts used heterologous growth hormone genes andpromoters and failed to produce these fish (e.g. Chourrout et al., 1986;Penman et al., 1990; Brem et al., 1988; Gross et al., 1992), enhancedgrowth of transgenic fish has been demonstrated in several fish speciesincluding Atlantic salmon, several species of Pacific salmons, and loach(e.g. Du et al., 1992; Delvin et al., 1994, 1995; Tsai et al., 1995).

The tiger barb (Puntius tetrazona, or, historically, Barbus tetrazona,syn. Capoeta tetrazona, also known commonly as the Sumatra barb),originally from Asia (more specifically, Malaysia, Indonesia, andBorneo) has been commercially cultured in the United States at least asearly as 1950 (Innes, 1950). The species name “tetrazona” refers to thefour vertical stripes seen on the wild-type tiger barb. However, for theornamental fish industry the dark striped pigmentation of the adulttiger barb does not aid in the efficient display of various colors. Thealbino tiger barb, or “albino barb” is a variant that arose duringdomestication and shows decreased pigmentation. The availability of suchfish having modified pigmentation for transgenesis with fluorescentproteins would result in better products for the ornamental fishindustry due to better visualization of the various colors.

Many fluorescent proteins are known in the art and have been used toinvestigate various cellular processes, including fluorescent proteinsexhibiting various green, red, yellow, blue, or purple colors. Althoughtransgenic experiments involving fluorescent proteins have provided newmarkers and reporters for transgenesis, progress in the field ofdeveloping and producing ornamental fish that express such proteins hasbeen limited.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention concerns making transgenicfluorescent fish and providing such fish to the ornamental fishindustry.

In some embodiments, transgenic fish or methods of making transgenicfish are provided. In certain aspects, the transgenic fish are fertile,transgenic, fluorescent fish. In a particular embodiment, the fish foruse with the disclosed constructs and methods is the albino barb. Barbskin color is determined by pigment cells in their skin, which containpigment granules called melanosomes (black or brown color), xanthosomes(yellow color), erythrosomes (orange or red color), or iridosomes(iridescent colors, including white color). The number, size, anddensity of the pigment granules per pigment cell influence the color ofthe fish skin. Albino barb have diminished number, size, and density ofmelanosomes and hence have lighter skin when compared to the wild typetiger barb.

In certain specific embodiments there are provided transgenic barbs orprogeny thereof comprising specific transgenic integration events,referred to herein as transformation events. These fish are ofparticular interest because, for example, they embody an aestheticallypleasing green color. Transgenic fish comprising these specifictransgenic events may be homozygous or heterozygous (including, forexample, hemizygous) for the transformation event. Homozygous fish bredwith fish lacking a transformation event will in nearly all casesproduce 100% heterozygous offspring. Eggs, sperm, and embryos comprisingthese specific transgenic events are also included as part of theinvention.

In one such embodiment regarding a specific transgenic integrationevent, a green transgenic barb or progeny thereof is provided comprisingchromosomally integrated transgenes, wherein the barb comprises the“Green barb 1 transformation event,” sperm comprising the Green barb 1transformation event having been deposited as ECACC accession no.12103003. The chromosomally integrated transgenes may be present on oneintegrated expression cassette or two or more integrated expressioncassettes. In certain aspects, such a transgenic barb is a fertile,transgenic barb. In more specific aspects, such a barb is a transgenicalbino barb. Such a transgenic barb may be homozygous or heterozygous(including, for example, hemizygous) for the transgenes or integratedexpression cassette(s).

Also disclosed are methods of providing a transgenic barb comprising theGreen barb 1 transformation event to the ornamental fish market. In someembodiments, the method comprises obtaining a transgenic barb comprisingchromosomally integrated transgenes, wherein the barb comprises the“Green barb 1 transformation event,” sperm comprising the Green barb 1transformation event having been deposited as ECACC accession no.12103003, and distributing the fish to the ornamental fish market. Suchfish may be distributed by a grower to a commercial distributor, or suchfish may be distributed by a grower or a commercial distributor to aretailer such as, for example, a multi-product retailer having anornamental fish department.

In some aspects, methods of producing a transgenic barb are providedcomprising: (a) obtaining a barb that exhibits fluorescence andcomprises one or more chromosomally integrated transgenes or expressioncassettes, wherein the barb comprises the “Green barb 1 transformationevent,” sperm comprising the Green barb 1 transformation event havingbeen deposited as ECACC accession no. 12103003; and (b) breeding theobtained barb with a second barb to provide a transgenic barb comprisingthe Green barb 1 transformation event. The second barb may be atransgenic or non-transgenic barb.

In further embodiments, also provided are methods of producing atransgenic organism, the method comprising using sperm comprising theGreen barb 1 transformation, such sperm having been deposited as ECACCaccession no. 12103003, to produce transgenic offspring. Such offspringmay be, for example, a barb, a species of the Puntius genus, a fishspecies or genus related to barb, or another fish species or genus. Insome aspects, the fish may be produced using in vitro fertilizationtechniques known in the art or described herein.

As used in this specification, “a” or “an” may mean one or more. As usedherein in the claim(s), when used in conjunction with the word“comprising,” the words “a” or “an” may mean one or more than one.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.” As used herein “another”may mean at least a second or more.

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

Any embodiment of any of the present methods, kits, and compositions mayconsist of or consist essentially of—rather thancomprise/include/contain/have—the described features and/or steps. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

DETAILED DESCRIPTION OF THE INVENTION Transgenic Fish

In some aspects, the invention regards transgenic fish. Methods ofmaking transgenic fish are described in, for example, U.S. Pat. Nos.7,135,613; 7,700,825; 7,834,239, each of which is incorporated byreference in its entirety.

It is preferred that fish belonging to species and varieties of fish ofcommercial value, particularly commercial value within the ornamentalfish industry, be used. Such fish include but are not limited tocatfish, zebrafish, medaka, carp, tilapia, goldfish, tetras, barbs,sharks (family cyprinidae), angelfish, loach, koi, glassfish, catfish,discus, eel, tetra, goby, gourami, guppy, Xiphophorus, hatchet fish,Molly fish, pangasius. A particular fish for use in the context of theinvention is a barb, Puntius tetrazona. Barb are increasingly popularornamental animals and would be of added commercial value in variouscolors. Barb embryos are easily accessible and nearly transparent. Afish that is of particular use with the disclosed constructs and methodsis the Albino Barb. Barb skin color is determined by pigment cells inthe skin, which contain pigment granules called melanosomes. The number,size, and density of the melanosomes per pigment cell influence thecolor of the fish skin. Albino Barb have diminished number, size, anddensity of melanosomes and hence have lighter skin when compared to thewild type barb.

Fertilization from Frozen Sperm

Fish sperm freezing methods are well-known in the art; see, e.g., Walkerand Streisinger (1983) and Draper and Moens (2007), both of which areincorporated herein by reference in their entireties. To obtaintransgenic fish disclosed herein, frozen barb sperm may be used tofertilize eggs

Briefly, one or two breeding pairs of barb should be placed in a shoeboxwith an artificial spawning mat. The water level in the shoebox shouldbe ˜2-3 inches and kept at 75-85° F. Low salinity (conductivity 100-200uS/cm) and slight acidity (˜pH 6.9) promote spawning. The fish may beexposed to a natural or artificial light cycle; the photoperiod startsat 8 am and ends at 10 pm. The following morning, remove and discard theeggs. Barb may be anesthetized by immersion in tricaine solution at 16mg/100 mL water. After gill movement has slowed, remove one female,rinse it in water, and gently blot the belly damp-dry with a papertowel. The eggs should not be exposed to water as this will preventfertilization. Gently squeeze out the eggs onto a slightly concavesurface by applying light pressure to the sides of the abdomen with athumb and index finger and sliding the fingers to the genital pore.Ready to spawn females will release the eggs extremely easily, and careshould be taken not to squeeze the eggs out while blotting the fish.Good eggs are yellowish and translucent; eggs that have remained in thefemale too long appear white and opaque. The females will release theeggs only for an hour or so. Eggs from several females may be pooled;the eggs can be kept unfertilized for several minutes. The sperm isthawed at 33° C. in a water bath for 18-20 seconds. 70 μl roomtemperature Hanks solution is added to the vial and mixed. The sperm isthen immediately added to the eggs and gently mixed. The sperm and eggsare activated by adding 750 μl of fish water and mixing. The mixture isincubated for 5 minutes at room temperature. The dish is then filledwith fish water and incubated at 28° C. After 2-3 hours, fertile embryosare transferred to small dishes where they are further cultured.

Parichy and Johnson, 2001, which is incorporated by reference in itsentirety, provides additional examples regarding in vitro fertilization.

The invention further encompasses progeny of a transgenic fishcontaining the Green barb 1 transformation event, as well as suchtransgenic fish derived from a transgenic fish egg, sperm cell, embryo,or other cell containing a genomically integrated transgenic construct.“Progeny,” as the term is used herein, can result from breeding twotransgenic fish of the invention, or from breeding a first transgenicfish of the invention to a second fish that is not a transgenic fish ofthe invention. In the latter case, the second fish can, for example, bea wild-type fish, a specialized strain of fish, a mutant fish, oranother transgenic fish. The hybrid progeny of these matings have thebenefits of the transgene for fluorescence combined with the benefitsderived from these other lineages.

The simplest way to identify fish containing the Green barb 1transformation event is by visual inspection, as the fish in questionwould be green colored and immediately distinguishable fromnon-transgenic fish.

EXAMPLES

Certain embodiments of the invention are further described withreference to the following examples. These examples are intended to bemerely illustrative of the invention and are not intended to limit orrestrict the scope of the present invention in any way and should not beconstrued as providing conditions, parameters, reagents, or startingmaterials that must be utilized exclusively in order to practice the artof the present invention.

Example 1 Green Transgenic Barb

Transgenic fish exhibiting a green color are provided. The specifictransgenic events embodied in these fish are designated Green barb 1.Sperm from these fish may be used to fertilize barb eggs and therebybreed transgenic barb that comprise these specific transgenicintegration events. Sperm from this line will be deposited at theEuropean Collection of Cell Cultures (ECACC) as “Green barb 1”(accession no. 12103003).

The fluorescent transgenic fish have use as ornamental fish in themarket. Stably expressing transgenic lines can be developed by breedinga transgenic individual with a wild-type fish, mutant fish, or anothertransgenic fish. The desired transgenic fish can be distinguished fromnon-transgenic fish by observing the fish in white light, sunlight,ultraviolet light, blue light, or any other useful lighting conditionthat allows visualization of the green color of the transgenic fish.

The fluorescent transgenic fish should also be valuable in the marketfor scientific research tools because they can be used for embryonicstudies such as tracing cell lineage and cell migration. Additionally,these fish can be used to mark cells in genetic mosaic experiments andin fish cancer models.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the methods described herein without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope, and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 7,135,613-   U.S. Pat. No. 7,700,825-   U.S. Pat. No. 7,834,239-   Brem et al., Aquaculture, 68:209-219, 1988.-   Chourrout et al., Aquaculture, 51:143-150, 1986.-   Delvin et al., Nature, 371:209-210, 1994.-   Draper and Moens, In: The Zebrafish Book, 5^(th) Ed.; Eugene,    University of Oregon Press, 2007.-   Du et al., Bio/Technology, 10:176-181, 1992.-   Innes, W. T., Exotic Aquarium Fishes: A work of general reference,    Innes Publishing Company, Philadelphia, 1950.-   Gross et al., Aquaculture, 103:253-273, 1992.-   Khoo et al., Aquaculture, 107:1-19, 1992.-   Lamason et al., Science, 310(5755):1782-1786, 2005.-   Penman et al., Aquaculture, 85:35-50, 1990.-   Powers et al., Mol. Marine Biol. Biotechnol., 1:301-308, 1992.-   Sin et al., Aquaculture, 117:57-69, 1993.-   Szelei et al., Transgenic Res., 3:116-119, 1994.-   Tsai et al., Can. J. Fish Aquat. Sci., 52:776-787, 1995.-   Walker and Streisinger, Genetics 103: 125-136, 1983.-   Xu et al., DNA Cell Biol., 18, 85-95, 1999.-   Zelenin et al., FEBS Lett., 287(1-2):118-120, 1991.-   Zhu et al., Z. Angew. Ichthyol., 1:31-34, 1985.

What is claimed is:
 1. A transgenic barb comprising a chromosomallyintegrated expression cassette encoding a green fluorescent protein,wherein the integrated expression cassette encoding the greenfluorescent protein is designated as the “Green barb 1 transformationevent” comprised in the sperm deposited as ECACC accession no. 12103003.2. The transgenic barb of claim 1, further defined as a fertile,transgenic barb.
 3. The transgenic barb of claim 1, further defined as atransgenic Albino Barb.
 4. The transgenic barb of claim 1, wherein thetransgenic barb is homozygous for the integrated expression cassette. 5.The transgenic barb of claim 1, wherein the transgenic barb isheterozygous for the integrated expression cassette.
 6. A method ofproviding a transgenic barb to the ornamental fish market, comprisingobtaining the transgenic barb of claim 1, and distributing thetransgenic barb to the ornamental fish market.
 7. The method of claim 6,wherein the transgenic barb is distributed by a grower to a commercialdistributor.
 8. The method of claim 6, wherein the transgenic barb isdistributed by a grower or a commercial distributor to a retailer. 9.The method of claim 8, wherein the retailer is a multi-product retailerhaving an ornamental fish department.
 10. A method of producing atransgenic barb comprising: (a) obtaining a transgenic barb thatexhibits fluorescence and comprises a chromosomally integratedexpression cassette encoding a green fluorescent protein, wherein theintegrated expression cassette encoding the green fluorescent protein isdesignated as the “Green barb 1 transformation event” comprised in thesperm deposited as ECACC accession no. 12103003; and (b) breeding theobtained transgenic barb with a second barb to provide a transgenic barbcomprising the “Green barb 1 transformation event” comprised in thesperm deposited as ECACC accession no.
 12103003. 11. The method of claim10, wherein the second barb is a non-transgenic barb.
 12. A progeny ofthe transgenic barb of claim 1, wherein the progeny exhibitsfluorescence and comprises the integrated cassette encoding the greenfluorescent protein designated as the “Green barb 1 transformationevent” comprised in the sperm deposited as ECACC accession no. 12103003.13. The progeny transgenic barb of claim 12, further defined as afertile, transgenic barb.
 14. The progeny transgenic barb of claim 12,further defined as a transgenic Albino Barb.
 15. The progeny transgenicbarb of claim 12, wherein the progeny transgenic barb is homozygous forthe integrated expression cassette.
 16. The progeny transgenic barb ofclaim 12, wherein the progeny transgenic barb is heterozygous for theintegrated expression cassette.
 17. A method of providing a transgenicbarb to the ornamental fish market, comprising obtaining the progenytransgenic barb of claim 12, and distributing the progeny transgenicbarb to the ornamental fish market.
 18. The method of claim 17, whereinthe progeny transgenic barb is distributed by a grower to a commercialdistributor.
 19. The method of claim 17, wherein the progeny transgenicbarb is distributed by a grower or a commercial distributor to aretailer.
 20. The method of claim 19, wherein the retailer is amulti-product retailer having an ornamental fish department.
 21. Amethod of producing a transgenic barb comprising: (a) obtaining thetransgenic barb of claim 12; and (b) breeding the obtained transgenicbarb with a second barb to provide a transgenic barb comprising the“Green barb 1 transformation event” comprised in the sperm deposited asECACC accession no.
 12103003. 22. The method of claim 21, wherein thesecond barb is a non-transgenic barb.